Endowing lymphocytes with antibody specificity

ABSTRACT

There are produced recombinant gene pairs which endow mononuclear cells, mainly various lymphocyte type cells, with antibody-type specificity. In specific gene pairs the rearranged gene pairs code for a binding site of an antibody molecule from the same species, of the T-cell receptor gene, or another species. Gene pairs of the invention code, for example, for antibodies specific towards tumor-specific antigens, viral antigens, modified self antigens, bacterial or fungal antigens, autoimmune type disease antigens and the like. The invention further relates to expression vectors for the effective transfection of such cell types comprising such a recombinant gene pair, to methods for producing same and to pharmaceutical compositions comprising as active ingredient an effective quantity of lymphocytes transfected with such gene pairs.

The present application is a continuation of application Ser. No.07/505,277, filed Apr. 6, 1990, now abandoned, which was acontinuation-in-part of application Ser. No. 07/346,483, filed May 2,1989, now abandoned, the entire contents of both of which are herebyincorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to gene pairs of recombinant DNA, whichgene pairs are adapted to endow mononuclear cells with antibody-typespecificity. Various types of cells are suitable, for example,lymphocytes such as T-killer cells, T-helper cells, T-suppressor cells,lymphokine activated cells and any other cell type which expresses theCD3/TcR complex.

The gene pair consists of one gene which comprises a DNA sequencecorresponding to exons coding for the constant region of the cellreceptor, and another gene which comprises sequences corresponding tothe leader and rearranged variable and joining exons encoding a specificantibody's heavy and light chains.

The present invention further relates to suitable vectors fortransfecting cells of the type defined above with such gene pairs. Thecells may be transfected with a single vector bearing the gene pair, orwith two different vectors, each bearing one gene of the gene pairs.

The present invention further relates to cells of the type defined aboveinto which such gene pairs have been introduced so as to obtain theirexpression, and also to pharmaceutical prophylactic and curativecompositions containing an effective quantity of such cells.

In general terms, the present invention relates to a process for thegeneration of lymphocytes transfected with one or two expression vectorscontaining DNA encoding a chimeric T-cell receptor. As set out in thefollowing, there was constructed a model system which comprisesexpression vectors which were transfected and which were functionallyexpressed in T-killer cells, i.e., which directed the cellular immuneresponse of the lymphocyte against a predefined target antigen in anon-MHC restricted manner.

The recombinant lymphocyte cells of the present invention may be used innew therapeutic treatment processes. For example, T cells isolated froma patient may be transfected with DNA encoding a chimeric receptorincluding the variable region of an antibody directed toward a specificantigen, and then returned to the patient so that the cellular immuneresponse generated by such cells will be triggered by and directedtoward the specific antigen in a non-MHC restricted manner.

Because of the restrictions imposed by corecognition of self MHC plusantigen, the acquisition of new specificity by grafting of TcR genes islimited to inbred combinations. Such manipulations are practicallyimpossible in an outbred population.

BACKGROUND OF THE INVENTION

Unlike antibodies, the T cells recognize antigen only in associationwith products of the major histocompatibility complex (MHC). Such dualrecognition is mediated by combination of the variable regions of boththe α and β chains that comprise the antigen recognizing T-cell receptor(TcR). Recently it became possible to endow T cells with a givenspecificity by DNA mediated transfer of cloned genes coding for the αand β TcR chains (Dembic et al., Nature, 320, 232-238 (1986)). Ingeneral, the expression as a dimer of both α and β chains of a given TcRhas been required in order to display a defined specificity although ithas been implicated that the Vβ is responsible for the MHC specificity(Saito et al., Nature, 329, 256-259 (1987)).

Expression of a chimeric receptor composed of immunoglobulin-derived Vregions and T cell receptor-derived C regions has been achieved byKuwana et al, Bioch. Biophys. Res. Comm., 149, 960-968 (1987).Expression was achieved in helper T cells and the criterion forexpression was an increase in cytosolic calcium concentration. Anincrease in cytosolic calcium concentration, however, does not establishthat one will obtain the various functional activities characteristic ofa cellular immune response by helper cells or other lymphocytes.

SUMMARY OF THE INVENTION

In order to overcome the limitations set out above, and in order to beable to design at will T-cells having a desired predeterminedspecificity, there were constructed T-cells with antibody-typespecificity. The invention is applicable to a wide variety of cells asset out herein.

According to the present invention, it is possible to construct andfunctionally express chimeric TcR that recognize antigen in non-MHCrestricted manner, effectively transmit transmembrane signal for T cellactivation, and mediate effector cell functions such as lymphokinesecretion or killing of target cells.

Based on the extensive degree of similarity in structure andorganization between the antibody and TcR molecules, it was assumed thatit will be possible to replace the V-region of TcR with an antibody'sone in a manner that will result in a chimeric TcR. However, it was notpredictable that such chimeric TcR's would retain their T cell functionsso as to trigger or direct the cellular immune response of the cell.Even the results of Kuwana et al do not lead to such predictability,particularly in view of the following. First, it is known that there aretwo signals needed for the activation of T cells (Wagner et al, J. Exp.Med., 155, 1876 (1982)). Kuwana et al disclosed that the receptorexpression caused an increase in cytosolic calcium concentration.However, while calcium activation is one pathway leading to T cellactivation, there is also another pathway involving inositol phosphate.The rise of calcium alone is inadequate to cause IL-2 production andproliferation of T cells. Although ligand binding to the T cell receptorinitiates two early activation signals (calcium raised and PKCactivation) as reviewed in Weiss et al, Ann. Rev. Immunol., 4, 543(1986), they are not sufficient to cause IL-2 production andproliferation of T cells (Linch et al, Immunolo. Rev., 95, 138 (1987)).

Furthermore, killer cells involve different accessory molecules anddifferent target cell presenting antigens compared to helper cells,i.e., MHC Class 1 versus MHC Class 2. Indeed, prior to the presentinvention it was thought that binding of the receptor to the MHC antigentriggered the lytic action of killer cells; the present inventorsdiscovered that such binding was not necessary in order to triggerkiller function. This was certainly not predictable from the work ofKuwana et al.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph showing the degree of IL-2 production by G.2--one ofthe transfectants which received and express the chimeric TcR (cTcR)genes.

FIG. 2 shows the analysis of the expression of chimeric polypeptideanalyzed by immunoblotting of cell lysates and by immunoprecipitation.

FIG. 3 shows cytotoxic activity of transfectants. The differenttransfectants and MD.45 parental cells were incubated at aneffector:target ratio of 2:1 with ⁵¹ Cr-labeled target cells. Effectorcells were used either without stimulation or following preincubationwith irradiated EL4 or TNP-A.20 stimulator cells.

FIG. 4 shows stimulation of Jurkat cells expressing the chimeric-TcR byTNP-modified (closed circle) or non-modified (open circle) cells. IL-2production following 24 hr stimulation was determined by theproliferation of CTL-L cell line using the MTT calorimetric assay.

FIG. 5 shows TNP specific activity of transfected murine T cell lines.The ability of OE4 cells transfected by cTcR to lyse various targetcells was estimated by the ⁵¹ Cr killing assay (A). The ability ofvarious stimuli to induce proliferation of OD1 transfected cells wasdetermined by the MTT assay (B).

FIG. 6 shows expression of cTcR directed at human ovarian cell carcinomaon the surface of T cells transfected with the OVB3-cTcR.Immunofluorescence staining was performed using purified polyclonalrabbit anti-OVB3 mABs, and analyzed by the FACS IV. Control staininggave 2 peaks the first negative, the second due to Fc receptor bearing Tcells.

FIG. 7 shows the oligonucleotides serving as universal consensus primersfor J_(H) and J_(K).

FIG. 8 shows the shuttle cassette vectors used to express thechimeric-TcR genes.

FIG. 9 describes the procedure used to amplify and clone V_(H) and V_(L)using the "Anchored PCR" technique.

FIG. 10 shows cRNA products of mRNA amplified from a hybridoma. Probesused in the Southern analysis: (dc) (middle) and Universal J_(H) andJ_(K) primers (lower).

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

According to the present invention, there were constructed chimeric Tcell receptor genes by recombining the V_(H) and V_(L) gene segments ofan anti-TNP antibody with the constant region exons of the T cellreceptor's (TcR) α and β chain. Following transfection into cytotoxichybridomas, expression of a novel functional T cell receptor wasdetected. The chimeric receptor manifested the idiotype of the antibodyand endowed the T cells with non-MHC restricted, anti-TNP reactivity.This model system demonstrates that chimeric TcR with an antibody-likebinding site can be designed and functionally expressed in a variety ofT-cells. By means of this manipulation, T-cells of any desiredspecificity may be engineered at will, provided that such specificitycan be predefined by monoclonal antibodies.

The various aspects of the present invention are suitable gene pairs forintroduction and expression in certain vectors and the use of suchvectors for transfecting cells (T-cells and others, as defined) in orderto endow them with a predetermined antigenic specificity. The inventionfurther relates to pharmaceutical compositions for the prevention andcure of certain diseases. Other aspects will become clear hereinafter.

As stated above, the present invention relates to lymphocytes,comprising populations of T-killer cells, T-helper cells, T-suppressorcells, lymphokine activated cells, and any other type of cell whichexpresses CD3/TcR. As further stated above, the present inventionrelates to a recombinant gene-pair adapted to endow mononuclearlymphocyte cells with antibody-type specificity, where the genes are:

a. DNA coding for the constant region of a lymphocyte receptor, andpreferably genomic segments containing exons coding for the constantregion of the T-cell receptor (α, β, γ, δ, chains), and

b. DNA coding for the heavy and light chains of an antibody specific fora predefined antigen, preferably gene-segments containing the leader andrearranged variable+joining exons encoding for a specific antibody'sheavy and light chains. The mononuclear lymphocyte cells are preferablypopulations of T-killer cells, T-helper cells, T-suppressor cells,lymphokine activated cells, and the like.

According to a preferred aspect, the present invention relates to a genepair with gene segments coding for the constant region of α or β chains,each of which being ligated with either the rearranged variable genesegment of the antibody's heavy or light chain, and vice versa. Itshould be understood that the T-cell receptor from which such constantregion is taken can be of the same species or of a different species asthe lymphocyte into which it is inserted. Preferably, the rearrangedgene segments code for a binding site of an antibody molecule eitherfrom the same species of the T-cell receptor gene or another species,where the rearranged gene segments code for antibody specificity towardstumor-specific antigens, tumor-associated antigens, viral antigens,modified self-antigens, parasitic antigens, bacterial antigens, fungalantigens, autoimmune disease type antigens, or other foreign antigens.

According to a further embodiment, the rearranged gene segments code formonoclonal antibodies reacting with a defined type of tumor cells orHIV-infected cells. The invention further relates to expression vectorsfor the efficient transfection of a cell type, comprising a recombinantgene pair as defined above. Such a vector can be a plasmid or retrovirusbackbone containing promoter, polyadenylation site and drug selectionmarkers. Preferred vectors according to the invention are the plasmidspRSV2 and PRSV3. There may be used a pair of expression vectors in whichone vector comprises a plasmid with one selection marker (such asneo^(R)) into which a rearranged gene segment coding for the variableregion of the antibody's light chain together with either the genesegment coding for the constant region of the T-cell receptors α or βchains are cloned, and the second vector comprises another selectionmarker (such as gpt) into which a rearranged gene segment coding for thevariable region of the antibody's heavy chain together with a genesegment of the constant region of the complementary T-cell receptor'schain (β or α) used in the first vector. Alternatively, DNAcorresponding to both chimeric genes, along with a single drug selectionmarker, may be put on a single vector for transfection into the hostcells.

The present invention further relates to mononuclear lymphocyte cellscontaining a chimeric pair of genes as defined above.

Yet another aspect of the present invention relates to a composition fortreating a patient in need thereof comprising as active ingredientlymphocytes which have been taken from the patient, propagated in vitro,and then transfected with a gene pair as above described or with avector as defined above. Such composition is then re-administered to thepatient.

Yet another aspect of the present invention relates to a process forproducing expression vectors according to the present invention and forconstructing a gene pair which comprises selecting a hybridoma producinga monoclonal antibody for the desired antigen, constructing a genomic orcDNA library from the restricted DNA fragments or RNA that contain therearranged V_(L) and V_(H) exons, isolating the clones carrying fulllength rearranged V_(L) and V_(H) exons, isolating from them the DNAsegments containing the leader exon and the rearranged VDJ_(H) andVJ_(L) exons and subcloning each of them into an expression vectorcontaining either the neo^(R) or gpt selection marker, inserting intoeach of the same vectors, 3' to the VDJ_(H) or VJ_(L), either one of thegenomic or cDNA fragments containing all of the constant region exonsand 5' flanking untranslated regions of the T-cell receptor α, β, γor δchains isolated from embryonic DNA or cDNA library, resulting in a setof chimeric genes comprised of V_(H) Cα, V_(L) Cβ, V_(L) Cα, V_(H) Cβ,V_(H) Cγ, V_(L) Cγ, V_(H) Cδ, V_(L) Cδ, each of which is cloned in oneof the expression vectors containing either the neo^(R) or gpt selectionmarkers.

Another aspect of the present invention is, in the process of joining avector (e.g., plasmid or retroviral) to DNA sequences coding for anantibody variable region and to DNA sequences coding for the constantregion of a T cell receptor, the improvement wherein the polymerasechain reaction is used to amplify mRNA sequences coding for the variableregion into DNA sequences coding for the variable region.

A key element in this innovation is the construction of chimeric Ab/TcRgenes. Until recently it involved cloning of the genes encoding theheavy (H) and light (L) chains of the antibody, isolation of the genesegments encoding the rearranged VDJ_(H) and VJ_(L), splicing them tothe TcR's Cα and Cβ genes and cloning the chimeric genes into vectorsthat allow their expression in T cells. These procedures are lengthy andrequire special conditions for each antibody.

We have recently developed a modified protocol that enables thegeneralization of the procedures to any antibody, and facilitates,quickens and simplifies the genetic engineering manipulations involvedin the construction of the cTcR genes. Practically, these modificationsare based on two major techniques we have recently developed.

1) Amplification of rearranged V_(H) and V_(L) genes directly from mRNAof any antibody forming cell, using the polymerase chain reaction (PCR)and primers composed of consensus sequences of J_(H) and J_(L) for the3' end and poly (dG) tail for the 5' end.

2) Cloning of the amplified (V_(H) and V_(L)) genes directly intoexpression vectors, containing the Cα and Cβ of the TcR.

1) Cloning of V_(H) and V_(L) by PCR

Oligonucleotide primers are used (FIG. 7) that correspond to the 3' endsof the joining (J) gene segment of the immunoglobulin H and L chain witha 3' donor splice signal (FIG. 7). The sequences of these primers wereadopted according to (11) to fit consensus sequences common to allmurine J_(H) and J_(L) and the splice sequence was added to allowsplicing to the first exon of the genomic TcR constant region. mRNA isprepared from the antibody forming cells (hybridomas or B cells) and acDNA strand is generated, using reverse transcriptase and one of theprimers. A short poly (dG) tail is added, 5' to this strand, using theenzyme terminal deoxynucleotydyl transferase. An oligo (dC) primer thatcontains a rare restriction site (Not I, FIG. 7) is now added as thesecond primer and the PCR is carried on to amplify the gene segmentbetween the two primers (12,13). The PCR product can be directly clonedinto the expression cassette described below. The rare restriction siteassists to choose clones with the right orientation.

2) Expression cassette for cTcR

The basic components of the expression cassette are the vectors pRSV2neoand pRSV3gpt (FIG. 8), which we have described before (3) that containgenes that allow for selection against neomycin (G418) and mycophenolicacid, respectively, eukaryotic transcription control elements derivedfrom the long terminal repeat (LTR) of Rous sarcoma virus, and a uniqueBamHI cloning site. Into this site were cloned genomic BamHI fragmentscontaining the constant region genes of either TcR α or β chain in theright transcriptional orientation. The 3' resulting BamHI site wasconverted, by partial BamHI digestion following by Klenow treatment,into a unique ClaI site that can be used for either linearization offinal constructs, or cloning another gene of interest. The 5' BamHI siteis used for cloning the amplified V_(H) or V_(L) genes described above.

FIG. 9 describes schematically the procedure in which we use the PCR toamplify mRNA from hybridoma producing anti-IgE mAbs, using V_(H) andV_(L) primers for reverse transcriptase cDNA synthesis. Followingseparation of the cDNA using spermin, oligo (dG) tail was added, usingterminal deoxynucleotydyl transferase and PCR amplification was carriedout using oligo (dC) as a 5' end primer. The products of this reactionare described in FIG. 10. The 0.5 Kb band in lane 2 represents the V_(H)fragment and the 0.5 Kb band in lane 3 represents V_(L) gene segment.The upper part shows the EtBr stained gel, the middle and lower parts ofFIG. 10 describe Southern analysis, using ³² P-V_(H) probe and ³²P-V_(L) probe correspondingly.

One aspect of the present invention relates to a process where acombination of two plasmids is used for the transfection of the T-cell,one of which comprises the variable of the light chain with either theconstant region of α or β; the other the variable of the heavy chainwith either of the Cα or Cβ. In another aspect, both regions are presentin the same plasmid or other vector.

The following is a description of a model experiment which demonstratesthe feasibility of the above general principles and its wide scope ofapplicability. The invention is not restricted to this specificembodiment.

To construct the chimeric TcR genes we ligated genomic segments each onecontaining the rearranged VJ and leader exons of either heavy or lightchain of the Sp6 anti-TNP, IgM antibody (Ochi et al.,Proc.Natl.Acad.Sci.USA, 80, 6351-6355 (1983)) with constant region exonsof either the α or β chains of the TcR. The chimeric genes were insertedinto pRSV based expression vectors containing the Rous sarcoma promotorand either the neo^(R) or the gpt drug resistance genes. By protoplastfusion each of the vectors were transfected into MD.45--a CTL hybridomaof BALB/c origin that can be stimulated by H-2D^(b) cells both for IL-2production and specific killing of target cells (Kaufmann et al.,Proc.Natl.Acad.Sci.USA, 78, 2502-2507 (1981)). Out of the drug resistanttransfectants, cells were selected that transcribed the chimeric gene(using V_(H) and V_(L) probes). The clone producing the highest levelsof one chain, was retransfected with the construct containing thecomplementary chain and the other drug marker. Double transfectants thatgrew in the presence of both mycophenolic acid and G.418 were checked byNorthern analysis for the transcription of both chimeric genes and byimmunoblotting and immunoprecipitation for the expression of the Sp6idiotype (using the 20.5 mAb). The functional expression of the chimericreceptor was evaluated by the ability of the cells to respond by IL-2production to TNPylated cells of various origins and by TNP-proteinantigens either alone or presented by different cells.

Following DNA transfer of the chimeric genes combination of either V_(L)Cα+V_(H) Cβ or V_(L) Cβ+V_(H) Cα into the MD.45 CTL hybridoma, almostall the transfectants transcribed both complementary chains of 1.8 Kbfor V_(L) Cα or β and 1.9 Kb for V_(H) Cα and β chains (Gross et al.,Proc.Natl.Acad.Sci.USA, 86, 10024 (1989)). The expression of thechimeric polypeptide was analyzed by immunoblotting of cell lysatesusing anti-Sp6 idiotype mAb 20.5 or by immunoprecipitation using the20.5 mAb and anti-TcR β8.3 subgroup F23.1 mAb (FIG. 2). Undernon-reducing conditions the anti-id and antiβ TcR reacted with a broadband of apparent molecular weight of 80 Kd that is composed of two bandsof 83 Kd and 77 Kd. In some transfectants a band of 42-45 Kd was alsoapparent. After reduction, however, the idiotypic determinant recognizedby 20.5 mAb in the immunoblot was destroyed and the 80 Kd complex wasdissociated into the 42 Kd polypeptide. Interestingly, transfectantsthat received either the V_(H) Cα or V_(H) Cβ alone also expressed the83 Kd complex as well as the 42 Kd chain carrying the 20.5 idiotype.Considering that Md.45 hybridoma expresses its αβ dimer (Becker et al.,Nature, 316, 606-619 (1985)) and only the β chain of BW 5147 fusionpartner (Lustgarten and Eshhar, unpublished data), together with thefact that the 20.5 idiotype (as well as the anti-TNP binding site) isexpressed solely on V_(H) Sp6, we can conclude that in transfectantsthat had received the V_(H) Cα or V_(H) Cβ gene, a chimeric chain isexpressed that can form functional dimer with the autologouscomplementary α or β TcR chains. The chains in part of the dimers arenot linked by disulfide bond and therefore migrate as single chain inSDS-PAGE. The double transfectants most likely express on their surfacein addition to the V_(L) Cα-V_(H) Cβ (or V_(L) Cβ-V_(H) Cα) chimericreceptor dimers, also the heterodimers that result from variouscombinations of pairing of the chimeric chain with the complementaryendogenous α and β chains. These result in the broad band observed inthe immunoprecipitation of surface iodinated TcR by either anti-id oranti-TcR (FIG. 2).

In order to study whether the chimeric receptor preserved the antibody'santi-TNP specificity and the ability to transmit transmembrane signalfor T cell activation, we coincubated the transfectants with differentstimulator cells either TNPylated or in the presence of variousTNP-protein antigens. FIG. 1 shows the degree of IL-2 production byG.2--one of the transfectant that received V_(L) Cβ+V_(H) Cα chimericgenes. The transfected cells expressed on their surface both theendogenous TcR as evidenced by their reactivity (like the parental MD.45hybridoma) toward EL-4 stimulator cells. Unlike MD.45, they underwenttriggering by TNP covalently coupled to A.20 (BALB/c B lymphoma) orUC.29 (human B lymphoblastoid) and other TNP modified cells. In additionTNPylated proteins (such as TNP-BSA, TNP-KLH and others), couldstimulate IL-2 production by G.2 either when immobilized on plastic andeven better in the presence of BALB/c spleen cells or A.20 cells thatare known to be good antigen presenters. Interestingly, thetransfectants that received only the V_(H) Cα or the V_(H) Cβ chimericgene and expressed the Sp6 idiotype, could also respond to TNPindicating that the V_(H) of Sp6 contains all the information needed toconstruct the TNP-binding site.

Another manifestation of the functional expression of the chimericreceptor in the effector phase of T-cell response was demonstrated bythe ability of the transfectants to specifically kill haptenated targetcells as measured by the ⁵¹ Cr-release assay.

For one set of ⁵¹ Cr-release assay experiments, in the first round oftransfections with single chimeric genes, out of 48 cells seeded foreach transfection, growth was seen in 21 of those that received V_(L) Cα(termed GTA.a), 15 that received V_(L) Cβ (termed GTA.b), 9 thatreceived V_(H) Cα (termed GTA.c), and 13 that received V_(H) Cβ (termedGTA.d). Clones expressing high RNA levels of each series were thentransfected with the complementary construct. In all, out of 3×10⁷ cellstransfected, 54 independent transfectants were obtained from GTA.a thatreceived the V_(H) Cβ construct (termed GTA.e), 13 from GTA.b thatreceived V_(H) Cα (termed GTA.f), 10 from GTA.c that received V_(L) Cβ(termed GTA.g), and 18 from GTA.d that received V_(L) Cα (termed GTA.h).

In the ⁵¹ Cr-release assay (9), target cells were modified by TNP using10 mM 2,4,6-trinitrobenzenesulfonic acid as described (10). Killingassays were performed at different ratios of effector to ⁵¹ Cr-labeledtarget cells for 4-8 hr. As shown in FIG. 3, all cells studied (exceptGTA.g2, which lost its ability to recognize EL-4 cells) killed EL-4 (theH-2^(b) target cell of the MD.45 hybridoma) as well as TNP-EL4 cells andtheir lytic ability was increased following prestimulation with EL-4.However, only the transfectants could kill the TNP-A.20 target cells.Accordingly, stimulation with TNP-A.20 cells enhanced only the cytolyticreactivity of cells that expressed the chimeric TcR. Similar resultshave been obtained when TNP-458,H-2^(S) B-lymphoma cells were used astargets in the killing assay. These studies are compatible with the ideathat the chimeric receptor can mediate non-MHC restricted,antigen-specific target cell lysis.

We attempted to transfect the V_(H) Cα+V_(L) Cβ chimeric genes encodingfor the Sp6 anti-TNP variable region into various T cell clones andtumor lines. The following T cells were used for transfection: OE4 is ananti-H-2^(d) allospecific cytotoxic T cell line (4). OD1 is an ovalbuminspecific, I-A^(d) restricted helper T cell line (5). Jurkat is a CD4⁺human T cell tumor. As target cells served: A.20, a BALB/c B cell line;NSO, a BALB/c myeloma/; RBL, a rat basophil leukemia cell/; and K562, ahuman myeloid cell line. pRSV2neoV_(H) Cα and pRSV3gptV_(L) Cβ (3) werelinearized by cleavage at a unique restriction site in the non-codingregion of the vectors. About 20 μg DNA were used to transfect 20×10⁶cells either by electroporation (6) or lipofectin (7). Followingtransfection, cells were allowed to recover in non-selective culturemedium. Conditioned medium and specific antigen presenting cells wereadded to the murine cell lines. After recovery of the cultured cells,selective drugs (G418 or mycophenolic acid) were added in the minimalconcentrations that were found to inhibit the growth of non-transfectedcells. Following 4-5 weeks of selection, growing cells were cloned andsub-cloned by limiting dilutions.

In the first set of experiments, we transfected the Jurkat human T cellline by electroporation. Following transfection the cells weredistributed at a concentration of 10⁵ /ml/cell in regular growth medium.48 hours later, G418 and mycophenolic acid were added. Cell growth wasapparent in about 60% of the wells, 2-3 weeks after transfection in thepresence of selective medium. More than half of the transfectantstranscribed the V_(H) Cα and could be stimulated by TNP-A.20 cells toyield IL-2. However, the most active transfectants, exemplified by cloneJCB6 (FIG. 4) which transcribed both chimeric chains. As shown in FIG. 4this transfectant could undergo stimulation by TNP-modified cells ofdifferent origin (human-K562, rat-RBL or mouse-A.20 and NSO).Non-TNPylated cells failed to trigger any response. Transfectants thatexpressed V_(L) Cβ alone did not display TNP specificity. As for theMD.45 hybridoma, the human Jurkat cells expressing both chimeric genes,displayed on their surface sufficient amounts of cTcR which could bedetected either by the anti-Sp6 idiotypic mAb 20.5 and by theanti-murine TcR mAb H.57. These studies indicate that cTcR genes, evenof murine origin, can associate with the human CD3 complex and beexpressed as functional receptors in human cells.

For practical application it is more crucial to study the expression ofthe chimeric receptor in T cell lines. Such cells are dependent fortheir replication on T cell growth factors, and are more susceptiblethan tumor cells or hybridomas. Indeed, the frequency of drug resistanttransfectants which we obtained after transfecting either the OE4cytotoxic murine T cell clone or the OD1 helper T cell clone was verylow. In fact, using either electroporation or the less traumaticlipofectin mediated transfection, we got only few drug resistant OE4cells and practically none of the OD1. We therefore have changed ourselection strategy by omitting the selective drugs and at different timeintervals following transfection, removed IL-2 from the growth mediumand replaced it with specific stimulus in the form of TNPylated targetcells. In preliminary experiments we could indeed induce TNP-specificproliferation of transfected cells. As can be seen in FIG. 5B, OD1 cellsfollowing transfection with the anti-TNP V_(H) Cα+V_(L) Cβ chimericgenes (OD1.CB) could proliferate in the presence of TNP-A.20. Asexpected, the addition of external IL-2 enhanced this TNP-specificproliferation. Non-TNPylated A.20 cells have no effect, and in the 24 hrassay period, IL-2 alone has only marginal effect. The cTcR genesconferred TNP specificity also on the cytotoxic OE4 cell line. OE4 is ofC57BL/6 origin and kills specifically H-2^(d) target cells, such as A.20(FIG. 5A). However, as shown in the figure, OE4.CB that wasco-transfected and selected for growth in the presence of both selectivedrugs, could kill TNP-EL4 (H-2^(b)) and not EL4 cells. Untransfected OE4cells failed to kill TNP-EL4 or EL4 cells. These are experiments inwhich we could demonstrate the expression of the cTcR by functionalassays. The yield of functional transfectants in the experimentsdescribed above was low. We recognize the transfection method as themost critical stage in determining the yield and level of expression ofthe cTcR genes. Transduction of genes with retroviral vectors have beenproved recently quite an effective method for DNA mediated gene transferinto human T cells (8).

In order to further establish the generality of the cTcR approach and toconstruct and express cTcR made of binding sites of anti-tumorantibodies in a functional manner, we have chosen two systems. The firstwas comprised of the murine B cell lymphoma 38C13 that express surfaceIgM. The idiotype of such IgM can serve as unique tumor cell antigen andwe used the SIC5 anti-38C13 id mAb (Moloney, D. G. et al., Hybridoma, 4,191, 1985), as the model to construct anti-tumor specific cTcR. Thesecond system was comprised of the monoclonal antibody OVB3 (Willingham,M. C. et al., Proc.Natl.Acad.Sci.(USA), 84, 2474, 1987) specific to theOVCAR, a human ovarian cell carcinoma which grows in tissue culture oras a tumor in nude mice.

Chimeric TcR genes were prepared from cloned V_(H) and V_(L) genes ofthe SIC5 and OVB3 mAbs basically as described herein or as detailed inGross, Waks and Eshhar, Proc.Natl.Acad.Sci.(USA), 86, 10024 (1989), theentire contents of which are hereby incorporated by reference. Fortransfection we used the cytotoxic T cell hybridoma MD45 or a TcRdeficient mutant (27J) which we isolated from the MD45 hybridoma.

As shown in Table I, demonstrating the activity of a transfectantreceiving the SIC5-based cTcR genes, the cTcR conferred on thetransfected T cell hybridoma specificity against the 38C13 lymphomatumor target cell. The transfectant underwent stimulation for IL-2secretion by 38C13 cells or 38C13 IgM when immobilized to plastic. Thespecificity, like that of the parental SIC5 mAb, was directed at the38C13 idiotype; hence, the mutant that lost the idiotype failed tostimulate. Therefore, as we showed before in the anti-TNP system, thecTcR conferred on the cells non-MHC restricted antibody typespecificity. Again, stimulation through the cTcR triggered the T cellhybridoma to its full activity.

In the OVB3 anti-human ovarian carcinoma cell system we could show thatT cell transfectants that received the OVB3-cTcR genes expressed ontheir surface the OVB3 idiotype (FIG. 6). These transfectants could bealso stimulated by the OVCAR tumor cells (not shown).

                  TABLE I    ______________________________________    Activity of T cells transfected with chimeric TcR genes composed    of antibody binding site specific to lymphoma surface idiotypes.sup.a.    Experiment              Stimulation  E/T     IL-2 Production    ______________________________________    I         38C13 cells  2:1     0.290                           9:1     0.300                           1:5     0.280              Id-mutant    1:1     0.025    II        Plastic Bound                           Soluble IL-2 Production              38C13 IgM    38C13                           (μg/ml)              -            --      0.010              +            --      0.245              +            50      0.001              +            5       0.015              +            0.5     0.152              +            0.005   0.235    ______________________________________     .sup.a CTL hybridoma, lacking the TcR chain were cotransfected with cTcR     genes composed of V.sub.H and V.sub.L of mAb SIC5 (anti38C13 id).     Following selections for growth in G418 and mycophenolic acid,     transfectants were analyzed for their ability to undergo stimulation with     the 38C13 lymphoma cells (Exp. 1) or plastic bound 38C13 IgM (Exp.II).     Soluble 38C13 IgM at various concentrations served as specific inhibitor     in the second  #experiment. As controls in both experiments, we used     either the cells or IgM of 38C13 mutant that does not bind the SIC5 mAb.     In both experiments stimulation was evaluated for 24 hr. The amount of IL     produced following stimulation was evaluated by its ability to support th     growth of CTLL line as measured by the MTT assay

Taken together, our results clearly demonstrate that it is possible toconstruct, transfect and functionally express chimeric T cell genes thatmanifest antibody specificity. This novel approach should be extended toenable the engineering at will of the specificity of T cells in non-MHCrestricted manner, in a way that a given set of genes could betransferred to T cells of any origin. Such T cells could then bereturned to their donor and manifest the acquired specificity. Followingsuch manipulation, the cells acquire a new specificity encoded by thechimeric genes that is of antibody-type, i.e., not restricted byself-MHC molecules.

The results obtained demonstrate that in a similar manner it is possibleto prepare a wide variety of pairs of such chimeric genes that aredirected at various target antigens which are predefined by specificmonoclonal antibodies. Such antigens can be those found in tumor cellsof a certain cancer, viral antigens, modified self antigens, antigens ofbacteria, parasites, fungi, antigens of autoimmune diseases, and anyother antigens toward which directing cellular immune responses canbenefit the patient. It is one of the advantages of this invention thatit enables taking the patient's own cells, their propagation in vitro,to select (if needed) a certain effector subpopulation (killers, helper,or suppressor cells), and to direct the desired specificity of suchcells by introducing into them the pair of engineered chimeric genes.Such cells, upon reimplantation into the patient, will function againstthe target antigens as dictated by the chimeric genes.

The foregoing description of the specific embodiments will so fullyreveal the general nature of the invention that others can, by applyingcurrent knowledge, readily modify and/or adapt for various applicationssuch specific embodiments without departing from the generic concept,and therefore such adaptations and modifications are intended to becomprehended within the meaning and range of equivalents of thedisclosed embodiments. It is to be understood that the phraseology orterminology herein is for the purpose of description and not oflimitation.

REFERENCES

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We claim:
 1. A mononuclear T-killer cell which expresses a chimericreceptor which permits said cell to selectively kill target cellsbearing a predefined target antigen in a non-MHC restricted manner, saidcell containing recombinant DNA encoding said chimeric receptor, saidDNA comprising:a. DNA coding for the two chains of the constant regionof a T-cell receptor (TcR), said two chains being the α and β chains orthe γ and δ chains, and b. DNA coding for the variable regions of theheavy and light chains of an antibody specific for the predefined targetantigen, such that, when expressed, one of said heavy and light chainsof said antibodies is attached to one of said two chains of said T-cellconstant region and the other of said heavy and light chains of saidantibodies is attached to the other of said two chains of said T-cellconstant regions.
 2. A mononuclear T-killer cell according to claim 1,wherein the cell into which said DNA is inserted is taken from a patientwho is to be treated with the modified T-killer cells.
 3. Apharmaceutical composition for treating a patient in need thereofcomprising as active ingredient an effective quantity of mononuclearT-killer cells in accordance with claim 1, and a suitable carrier oradjuvant.
 4. A pharmaceutical composition in accordance with claim 3,wherein the cell into which said DNA is inserted is taken from a patientwho is to be treated with the modified T-killer cells.
 5. A mononuclearT-killer cell in accordance with claim 1 that is a human cell.
 6. Amononuclear T-killer cell in accordance with claim 1, wherein:(a) saidDNA coding for the two chains of the constant region of a TcR aregenomic segments containing exons for said two chains; and (b) said DNAcoding for the variable regions of the heavy and light chains of anantibody specific for the predefined target antigen are gene-segmentscontaining the VDJ region leader, variable, and joining exons encoded insaid antibody's heavy and light chains.
 7. A mononuclear T-killer cellin accordance with claim 6, wherein said genomic segment (a) codes forthe constant region of TcR α and β chains.
 8. A mononuclear T-killercell in accordance with claim 1, wherein the TcR and the antibody fromwhich said DNA of (a) and (b), respectively, is obtained are of the samespecies.
 9. A mononuclear T-killer cell in accordance with claim 1,wherein the TcR and the antibody from which said DNA of (a) and (b),respectively, is obtained are of different species.
 10. A mononuclearT-killer cell in accordance with claim 1, wherein said heavy and lightchains are from an antibody specific towards a tumor-specific antigen, atumor-associated antigen, a viral antigen, a modified-self antigen, aparasitic antigen, a bacterial antigen, a fungal antigen or anautoimmune disease type antigen.
 11. A mononuclear T-killer cell inaccordance with claim 10, wherein said antibody is a monoclonal antibodyreacting with a defined type of tumor cell.
 12. A mononuclear lymphocytecell which expresses a chimeric receptor which permits said cell todirect its cellular immune response toward a predefined target antigenin a non-MHC restricted manner, said cell containing recombinant DNAencoding said chimeric receptor, said DNA comprising:a. DNA coding foran α, β, γ or δ chain of the constant region of a T-cell receptor (TcR),and b. DNA coding for the V_(H) region of an antibody specific for thepredefined target antigen, wherein said antibody is specific towards atumor specific antigen, a tumor associated antigen, a viral antigen, oran autoimmune disease type antigen.
 13. A mononuclear lymphocyte cell inaccordance with claim 12, wherein said DNA coding for an α, β, γ or δchain of the constant region of a TcR is DNA coding for an α chainthereof.
 14. A mononuclear T-killer cell in accordance with claim 12,wherein said DNA coding for an α, β, γ or δ chain of the constant regionof a TcR is DNA coding for an β chain thereof.
 15. A mononuclearT-killer cell in accordance with claim 12, wherein the TcR and theantibody from which said DNA of (a) and (b), respectively, is obtainedare of the same species.
 16. A mononuclear T-killer cell in accordancewith claim 12, wherein the TcR and the antibody from which said DNA of(a) and (b), respectively, is obtained are of different species.
 17. Amononuclear T-killer cell in accordance with claim 16, wherein saidantibody is a monoclonal antibody reacting with a defined type of tumorcell.
 18. A mononuclear T-killer cell according to claim 12, wherein thecell into which said DNA is inserted is taken from a patient who is tobe treated with the modified lymphocytes.
 19. A pharmaceuticalcomposition for treating a patient in need thereof comprising as activeingredient an effective quantity of mononuclear lymphocyte cells inaccordance with claim 12, and a suitable carrier or adjuvant.
 20. Apharmaceutical composition in accordance with claim 19, wherein the cellinto which said DNA is inserted is taken from a patient who is to betreated with the modified lymphocyte cells.
 21. A mononuclear lymphocytecell in accordance with claim 12 that is a human cell.
 22. A mononuclearlymphocyte cell in accordance with claim 12 that is a T-killer cell,wherein said chimeric receptor permits said cell to selectively killsuch target cells in a non-MHC restricted manner.
 23. A mononuclearlymphocyte cell which expresses a chimeric receptor which permits saidcell to direct its cellular immune response toward a predefined targetantigen in a non-MHC restricted manner, said cell containing recombinantDNA encoding said chimeric receptor, said DNA comprising:a. DNA codingfor the two chains of the constant region of a T-cell receptor (TcR),said two chains being the α and β chains or the γ and δ chains, and b.DNA coding for the variable regions of the heavy and light chains of anantibody specific for the predefined target antigen, such that, whenexpressed, one of said heavy and light chains of said antibody isattached to one of said two chains of said T-cell constant region andthe other of said heavy and light chains of said antibody is attached tothe other of said two chains of said T-cell constant regions, whereinsaid heavy and light chains are from an antibody specific towards tumorspecific antigens, tumor associated antigens, viral antigens, orautoimmune disease type antigens.
 24. A human mononuclear lymphocytecell which expresses a chimeric receptor which permits said cell todirect its cellular immune response toward a predefined target antigenin a non-MHC restricted manner, said cell containing recombinant DNAencoding said chimeric receptor, said DNA consisting essentially of:a.DNA coding for an α, β, γ or δ chain of the constant region of a T-cellreceptor (TcR), and b. DNA coding for the V_(H) region of an antibodyspecific for the predefined target antigen.
 25. A human mononuclearlymphocyte cell in accordance with claim 24, wherein said V_(H) regionis from an antibody specific towards a tumor-specific antigen, atumor-associated antigen, a viral antigen, a modified self-antigen, aparasitic antigen, a bacterial antigen, a fungal antigen or anautoimmune disease type antigen.
 26. A human mononuclear lymphocyte cellin accordance with claim 25, wherein said antibody is a monoclonalantibody reacting with a defined type of tumor cell.
 27. A humanmononuclear lymphocyte cell which expresses a chimeric receptor whichpermits said cell to direct its cellular immune response toward apredefined target antigen in a non-MHC restricted manner, said cellcontaining recombinant DNA encoding said chimeric receptor, said DNAcomprising:a. DNA coding for the two chains of the constant region of aT-cell receptor (TcR), said two chains being the α and β chains or the γand δ chains, and b. DNA coding for the variable regions of the heavyand light chains of an antibody specific for the predefined targetantigen, such that, when expressed, one of said heavy and light chainsof said antibody is attached to one of said two chains of said T-cellconstant region and the other of said heavy and light chains of saidantibody is attached to the other of said two chains of said T-cellconstant regions.
 28. A process of directing the immune response of apatient toward a predefined target antigen, comprising the steps of:(1)transfecting mononuclear lymphocyte cells with a recombinant DNAcomprising(a) DNA coding for an α, β, γ or δ chain of the constantregion of a T-cell receptor (TcR), and (b) DNA coding for the V_(H)region of an antibody specific for the predefined target antigen, and(2) inoculating the patient with the transfected lymphocyte cells.
 29. Aprocess in accordance with claim 28, wherein the mononuclear lymphocytecell is a T-killer cell.
 30. A process in accordance with claim 28,wherein the mononuclear lymphocyte cell is one which has been isolatedfrom the patient to be treated and wherein said inoculating stepcomprises reinoculating the transfected cell into the patient from whichit was isolated.
 31. A process in accordance with claim 30, wherein DNAencoding said V_(H) region is that of an antibody specific for a definedtype of tumor or HIV-infected cell.
 32. A process in accordance withclaim 28, wherein said DNA of (a) encodes the two chains of the constantregion of a TcR, said two chains being the α and β chains or the γ and δchains, and wherein said DNA of (b) encodes the variable regions of theheavy and light chains of an antibody specific for the predefined targetantigen, such that, when expressed, one of said heavy and light chainsof said antibody is attached to one of said two chains of said T-cellconstant regions and the other of said heavy and light chains of saidantibody is attached to the other of said two chains of said T-cellconstant regions.
 33. A process for producing a vector containing a DNAsequence coding for an antibody variable region and a DNA sequencecoding for the constant region of a T-cell receptor (TcR),comprising:preparing a TcR expression cassette containing regulatoryregions and the DNA encoding the constant region of a TcR; amplifying bypolymerase chain reaction an antibody variable region of known orunknown sequence using primers composed of consensus sequences of J_(H)or J_(C) for the 3' end and poly(dG) tail for the 5' end; and cloningthe amplified antibody variable regions directly into said TcRexpression cassette.